Generally for culturing microorganisms, two types of medium are in common use; liquid medium in which the microorganisms settle on the bottom surface of the culture dish, and semi-solid medium in which the microorganisms are evenly dispersed throughout the culture medium. Two configurations of light microscopes are in common use: the upright and the inverted microscope. The upright microscope has its objective lens above the specimen and is generally used to observe specimens placed on microscope slides, whereas the inverted microscope has its objective lens situated below the specimen and is generally used to observe microorganisms growing in culture. In order to obtain useful information from practically all types of renewing and mature cell populations, it is often desirable to observe living cell cultures in liquid medium, using an inverted microscope, or to observe living cell cultures in semi-solid medium, using an upright microscope. One use of this type of information and observation is in the treatment of cancer, where the efficacy of anti-cancer drugs on a particular patient's cancer cells can be assessed in tissue culture prior to treatment. Another use of this type of observation is in basic biological research wherein cell division and function can be studied at the level of the single cell.
Cultures performed in semi-solid medium in conventional culture dishes have the following limitations:
1. Since the cells are suspended throughout the entire volume of the culture medium at varying heights, constant focus adjustment of the microscope is necessary during observation, making quantitive observations tedious and time consuming.
2. Many of the cells under observation are sufficiently far removed from the objective lens of the microscope, (even using an inverted microscope) that high objective lens power cannot be used for observation.
These limitations may be overcome by using liquid culture medium in conjunction with an inverted microscope for observation. In liquid culture all the cells settle to the bottom of the dish in a monolayer and can be focused upon from below even using high objective lens power. Since all the cells lie in the same plane, once the plane is focused upon, no additional focusing is necessary. However, cultures performed in liquid medium in conventional culture dishes have the following limitations:
1. Cells are continuously moved about the bottom surface by the motion of the liquid medium, due to standing wave and convection currents.
2. There is mass movement in the culture upon handling, and spillage occurs readily with only slight tilting of the dish.
3. A meniscus is formed at the edge of the liquid culture medium, distorting the highly focused light from the light source passing through the upper surface of the liquid, making high resolution observation of cells, particularly toward the outside edges of the dish, very difficult and unreliable. Furthermore, the increased thickness of medium in the area of the meniscus results in an increase of cell density at the edge of the culture chamber. This leads to a high cell density in a region that cannot be well observed, a situation that is far from ideal.
Presently, culturing in semi-solid medium is in more common use than culturing in liquid medium, because no dish has hitherto been available that has been able to overcome the physical instability of liquid medium and of the cells in that medium. It is an object of this invention to provide a tissue culture device suitable for the observation of living cells and organisms while being cultivated in semi-solid or liquid medium, suitable for use with both upright and inverted light microscopes.
Another object of this invention is to provide a tissue culture device which contains the culture in an almost totally enclosed volume holding the culture stable by providing minimum free surface areas, thus preventing cell aggregation in certain areas, and mass movement upon handling of the dish.
Another object of this invention is to provide a tissue culture device able to compensate for evaporative water loss from the culture during extended culture periods.
Yet another object of this invention is to provide a tissue culture device which allows a reasonable tolerance in volume of the medium required to fill the fixed volume culture chamber.
Another object of this invention is to provide a tissue culture device with no optical distortion due to meniscus near the edge of the culture region.
Another object of this invention is to provide a tissue culture device containing more than one identical culture chamber, each with the above features.
The present invention solves the above problems by stabilizing the liquid medium in an almost totally enclosed fixed volume. This is accomplished by placing a window in contact with the entire top surface of the culture chamber.
For ease of explanation, the following will refer to a culture device having only one culture chamber, each culture chamber of a multiple chamber device being identical to each other.
In order to observe cells being cultured in semi-solid medium in conventional culture dishes it has been found necessary to use an inverted microscope in order to be able to place the objective lens closer to the cells being observed and thus achieve higher objective lens power observation than would be possible with an upright microscope. The present invention incorporates a transparent cover window that is lowered down to and in contact with the top surface of the culture chamber to permit observation of culture cells using an upright microscope at the same objective lens power as an inverted microscope. This is of advantage since there are many more upright microscopes currently in use than inverted microscopes.
Cells cultured in semi-solid medium are susceptible to vibration-induced oscillations since the support used to solidify the medium forms a gel, which is very resilient and responds to vibrations in the environment. Distortion due to meniscus, and aggregation of cells near the edge of the culture chamber occurs in semi-solid cultures in conventional culture dishes, as it does in liquid cultures. The present invention solves these problems involving semi-solid cultures by imparting a high degree of stability to the semi-solid medium, the surface of the upper window in contact with the medium greatly diminishing the ability of the medium to vibrate.
Although the present invention offers particular advantages for the observation of liquid cultures using an inverted microscope and for the observation of semi-solid cultures using an upright microscope, it may also be used for the observation of semi-solid cultures with an inverted microscope and for the observation of liquid cultures with an upright microscope.